The present invention relates to a method of and arrangement for producing uniform, unilamellar so-called small lipid vesicles, particularly for conversion of lamellarly arranged lipids into lipid vesicles.
Methods and arrangement of the above mentioned general type are known in the art. Lipid structures such as lamellae are accommodated in a suspension fluid inside a container and subjected to an ultrasonic treatment in condition of a constant temperature. Lipid vesicles are utilized, for example, for medical therapy and for basic research purposes. Drugs or effectors which control the intracellular metabolism and having intracellular receptors and binding-sites need to penetrate into the cell from outside. Many effectors normally formed inside the cell can neither leave the cell nor the surrounding medium. The therapeutic use of the substances of this class makes necessary to provide a transport mechanism which allows the irreversible incorporation of these non-membrane-permeable substances into the cells, namely without any possibility to leave the cells after this process. Such a transport mechanism must be, moreover, as independent as possible from the effector to be incorporated, i.e. it allows incorporation of effectors of any type, on the one hand, and should be cell-specific, i.e., allows the incorporation of effectors only in predetermined cells, on the other hand. A transport system with the above mentioned properties is provided by lipid vesicles which can include different substances, such as enzymes, drugs, chelate-forming substances, hormones, cell-effectors, antigenes, antibodies, interferon inductors and genes. In the lipid vesicles, the solvent and the substance dissolved in the solvent are enclosed by phospholipid bilayer membrane. The lipid membrane has a thickness of typically 4 nm, and the vesicles can have a diameter from 25 to 120 nm. The size of the vesicles can be determined by the laser light scattering, ultracentrifugation, gel-filtration or electron scanning microscopy.
An important field of application of the lipid vesicles is the incorporation of inositolhexaphosphate (IHP) into red blood cells (RBC) in accordance with the method described by Y. C. Nicolau and K. Gersonde, one of the instant co-inventors, in U.S. Pat. No. 4,192,869, for reducing the oxygen affinity of the intracellular hemoglobin. It is known that during storage of blood preserves, the oxygen affinity of the hemoglobin in the red cells is continuously increased. Furthermore during certain diseases an increased oxygen affinity of the hemoglobin can also be observed. This increased oxygen affinity is the reason that only a small portion of oxygen which is bound to the hemoglobin and circulated in the blood, is effectively released to the tissues. This high oxygen affinity of the hemoglobin can be reduced by binding certain effectors to the hemoglobin. The strongest effector of this type is the inositol hexaphosphate (IHP). The incorporation of IHP is attained in such a manner that the intact cells are incubated with IHP-loaded lipid vesicles and that by fusion of the lipid membrane of the cells and the vesicles IHP is incorporated into the cells where it is bound to hemoglobin and changes the oxygen affinity of the hemoglobin measurable by "right-shifting" of the hemoglobin-oxygen dissociation curve. After retransfusion of these IHP-loaded red blood cells into the blood vessels, a considerably greater portion of the oxygen stored in the red blood cells is released to the peripheral tissues. This property of the treated red blood cells is retained during the entire life of the cells.
For incorporation of the inositol hexaphosphate into red blood cells, small unilamellar IHP-loaded lipid vesicles with a diameter from 20 to 50 nm are required. It is known to produce lipid vesicles by disintegration of lipid suspensions in an ultrasonic field. The progress in the utilization of lipid vesicles was, however, very slow because the production of lipid vesicles suitable for fusion with the red blood cells in a satisfactory quantity is accompanied by considerable difficulties. The lipid vesicles suitable for this purpose must not only be produced in sufficient quantities, but also must be reproducible in identical sizes and thereby dosable in the therapeutic administration. The subsequent use of separation procedures for isolating suitable fractions of the particular lipid vesicles encounters many problems, for example maintaining of sterility and utilization of expensive and time-consuming separation techniques considerably reducing the biological effectiveness of the vesicles which have at room temperature a half-life of approximately one day. The only method which can produce large quantities of vesicles in short time is the ultrasonic disintegration technique.
In addition to the type and composition of the lipid vesicles, the success and the reproducibility of experimental work or of a therapeutic treatment, i.e. incorporation of IHP in red blood cells, depend essentially on the size of lipid vesicles. The verification, whether or not the disintegration of the lipid suspension produces lipid vesicles of sufficient homogeneity and thereby of good quality and in sufficient quantity, is generally performed in such a manner that with the produced lipid vesicles the desired IHP uptaken by red blood cells is controlled by chemically detecting the intracellular IHP, and by measuring the hemoglobin-oxygen dissociation curve of intact cells or by quantifying the desirable biological or therapeutic action of the IHP-loaded red blood cells in animal tests. The yield of IHP-loaden vesicles and the effectiveness of the treatment of red blood cells can only be evaluated after expensive and time-consuming experiments. The result of the ultrasonic preparation, namely the production of the vesicles suitable for fusion with red blood cells, can be evaluated only afterwards.
It has been shown that the production of lipid vesicles, particularly in large volumes, as required for therapeutic processes, with constantly maintained properties with the aid of the ultrasonic technique is difficult. Despite preservation of completely identical outer conditions during the disintegration of the lipid suspensions in the ultrasonic field, it was not possible to attain the uniform production of so-called small unilamellar and thereby fusion-active lipid vesicles.